We propose to continue the development of quantitative Fluorescent Speckle Microscopy (qFSM). qFSM is a variant of fluorescence microscopy to analyze the dynamics of subunits within macromolecular structures in living cells. In the first round of funding, we developed focus-stabilized TIRF FSM and spinning disk confocal imaging of actin cytoskeleton and focal adhesion dynamics in tissue culture cells, and developed image analysis software that converts the stochastic speckle image signal into high- resolution maps of the assembly, disassembly, and transport of the actin filaments. We exploited the quantitative information delivered by this technology to study the regulation of the actin cytoskeleton in migrating epithelial cells and thus established a new paradigm for how the cell builds specific actin-based structures to drive directed cell migration. Here, we seek to make the next critical steps towards our long term goal of establishing qFSM as a method for the comprehensive analysis of spatiotemporal dynamics and interaction of multiple macromolecular structures in living cells. Our specific aims are: Aim 1: To extend qFSM to the measurement of absolute rates of polymer turnover which will allow us to perform in situ biochemistry of actin dynamics in living cells. Aim 2: To extend qFSM to the analysis of microtubule and intermediate filament cytoskeletons. Aim 3: To establish correlational qFSM to probe the dynamic interaction between two or more macromolecular structures in living cells. These developments are motivated by the hypothesis-driven cell biological research in our labs on the fundamental mechanisms of cytoskeletal function in cell migration, but our technology is implemented generically with an eye towards its broad use by the biomedical research community. We propose to further develop the technique of quantitative Fluorescent Speckle Microscopy for the analysis of the dynamics and interaction of macromolecular assemblies. Our studies will focus on the spatial and temporal integration of the dynamics of the actin filament, microtubule, and intermediate filament cytoskeleton systems. These interactions are centrally implicated in a wide array of cell functions. Thus, the proposed developments will serve the cell biology community to investigate fundamental aspects of cell physiology and pathological behaviors.